A ceramidase is an enzyme which hydrolyzes a ceramide, a kind of a sphingolipid, into a sphingoid and a fatty acid. The sphingoid which is generated by hydrolyzing the ceramide with the ceramidase possesses various physiological activities such as inhibition of protein kinase C, activation of phospholipase D and inhibition of a calmodulin-dependent enzyme. As described above, the above-mentioned sphingoid is an important substance which is thought to be acting on the regulation of the cell functions because the sphingoid is involved in proliferation of cells and intracellular signal transduction. The ceramidase is an enzyme which plays an important role of the control of the amount of the above-mentioned sphingoid.
Ceramidases are classified into acidic ceramidases and neutral/alkaline ceramidases by the optimum pH. There have been, so far reported that the presence of a ceramidase possessing the optimum pH in an acidic range has been found in mammalian tissues such as rat brain [Biochemistry, 8, 1692-1698 (1969)], guinea pig epithelial cells [J. Biol. Chem., 270, 12677-12684 (1995)], human kidney [Biochim. Biophys. Acta, 398, 125-131 (1975)], spleen [Biochim. Biophys. Acta, 1004, 245-251 (1989)], fibroblasts [Biochem. J., 205, 419-425 (1982)], and epithelium [FEBS Lett., 268, 110-112 (1990)]; and human urine [J. Biol. Chem., 270, 11098-11102 (1995)], and the like.
In addition, it has been clarified that a bacterium belonging to the genus Pseudomonas produces a ceramidase, and this ceramidase is a ceramidase possessing optimum pH in an alkaline region [J. Biol. Chem., 273, 14368-14373 (1998)].
Among these ceramidases, amino acid sequences of the acidic ceramidase purified from human urine and nucleotide sequences of a gene encoding the ceramidase have been determined [J. Biol. Chem., 271, 33110-33115 (1996)]. In addition, an acidic ceramidase gene of a mouse has been obtained by utilizing its homology with the above-mentioned acidic ceramidase gene derived from human urine [Genomics, 50, 267-274 (1998)].
However, since all of the ceramidase genes derived from mammals which have been reported encode acidic ceramidases, amino acid sequences and genomic structures of the neutral/alkaline ceramidase in mammals have been completely unknown, so that biological functions of the neutral/alkaline ceramidase in higher organisms have not yet been elucidated at present.
In the studies on the elucidation of the in vivo functions of a ceramide, metabolic control therefor, diagnosis or treatment of a disease associated with the ceramide or the like, it is necessary to obtain detailed information concerning various enzymes associated with the ceramide, and the enzyme gene. However, as mentioned above, findings on the amino acid sequence and genes thereof of the neutral/alkaline ceramidase in mammals have not yet been obtained at present. Therefore, in order to develop the technique as described above pertaining to a ceramide, it is necessary to obtain some findings associated with a neutral/alkaline ceramidase, especially a gene thereof.
As mentioned above, although several reports have been made on cloning of ceramidase genes of a mammal, all of these reports are concerned with ceramidases possessing an activity in an acidic region, which cannot be expected to possess a homology with a ceramidase possessing an activity in a neutral alkaline region. Therefore, it has been difficult to obtain a neutral/alkaline ceramidase gene as a homolog of a nucleotide sequence of the acidic ceramidase gene.